Near infra-red labelling and tracking of corneal endothelial cells in-vivo

Following corneal transplantation, there is an initial, rapid decline in corneal endothelial cells (CECs) following surgery. Direct imaging of post-transplantation endothelial cells is only possible weeks after surgery and with a limited field of view. We have developed a labelling approach using 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide (DIR) dye solution, that enables tracking of labelled CECs in vivo for at least 1 month. Initial in vitro optimization, with assessments of dye concentration on fluorescence, cellular toxicity and cell migration, performed in propagated primary CECs. Subsequently, in vivo evaluation of cellular labelling was assessed within a rabbit wound healing model. Finally, real-time visualization of human cadaver donor tissue incubated in DIR transplanted into rabbits was achieved using a clinical confocal microscope. Results revealed detectable fluorescence increased with concentration to a plateau of 100 µg/ml, with no toxicity of CECs at any concentration evaluated. DIR-labelled CECs were detectable in vivo up to 1 month, and transplanted labelled donor graft could be visualized and were trackable in vivo. Acute endothelial rejection in 1 rabbit was evidenced by detectable DIR positive cells within the anterior chamber. DIR imaging allowed for detailed imaging of the transplanted human corneal endothelium, and enabled non-invasive observation of the corneal endothelial morphology following transplantation.


Scientific Reports
| (2022) 12:6338 | https://doi.org/10.1038/s41598-022-09677-w www.nature.com/scientificreports/ based on optical coherence tomography (OCT) imaging, are being developed 13 . Typically, only a small number of cells, between 30 to 100, are imaged at high magnification 14 . Image quality can be poor following endothelial keratoplasty (EK), especially with non-contact methods 15 . Poor image acquisition from off-axis portions of the cornea, and the lack of automated methods for stitching multiple images together, mean these methods do not allow for global assessment of the entire graft 16 . Consequently, tracking small areas of interest over time can be very difficult 17 . In both specular and confocal microscopy, image quality is reliant on a clear corneal stroma, meaning images obtained through oedematous or scarred corneas are often of poor quality. The presence of an intra-cameral gas bubble can also impact image quality. As such, it is often not possible to use these methods in the first 2 weeks following EK, nor is it possible to use these methods to examine endothelial healing following wounding studies, which result in significant swelling of the cornea 5 .
Wounding studies are commonly used to assess the efficacy of surgical/pharmaceutical interventions aimed at promoting endothelial healing [15][16][17] . In our previous studies, in vivo endothelial wound healing was assessed using repeated intracameral trypan blue injection 10 . Whilst this was a reliable and reproducible technique, it is an invasive procedure that carries the risk of damaging healing endothelial cells or introducing infections 18 . The method cannot be used in the early post-operative periods following EK or Descemet's membrane transplantation, whilst an intracameral gas bubble remains. Caution needs to be taken early after bubble resolution to avoid dislodging the recently transplanted material. An additional limitation of the trypan blue technique is that it only allowed global imaging of endothelial wound healing. This meant animals had to be sacrificed at staggered time intervals in order to perform histological evaluation of wound healing at a cellular level.
We previously developed and validated a method to image endothelial cells, in vivo, using Calcein AM; a green-fluorescent viability stain 5 . Whilst capable of global and cell-level imaging in the immediate post-operative period, our in vitro experiments showed that Calcein AM was rapidly cleared from endothelial cells indicating that any useful fluorescence signal will be extinguished by 24 h post-incubation. As such, it is not useful for longer-term tracking of endothelial cells in vivo. The fate of the transplanted cells can only truly be determined if methods to quantify transplanted corneal endothelial cell death immediately post-operatively can be combined with techniques to track endothelial cells through early post-operative phase, which we define as 1 month based on previous reports 19,20 .
In this study, we describe a technique for labelling and tracking corneal endothelial cells in vivo, both globally across the entire cornea and at an individual cell level. We compared these in vivo images to immuno-histology following animal sacrifice. We show this technique is suitable for tracking both native and transplanted endothelial cells and can be used in wound healing studies as well as xenotransplantation of human EK grafts into rabbits.

Results
Optimizing DIR concentration in vitro. Average DIR fluorescence increased with concentration in a linear-quadratic fashion. Comparison of average fluorescence levels at different doses, derived from macroscopic images taken from individual wells, revealed a significant difference between cells stained with 50 µg/ml and 100 µg/ml (repeated measures ANOVA, corrected p < 0.0001, N = 15 per group) but not between 100 and 200 µg/ ml (p = 0.78) (Fig. 1a). Based on these findings a dose of 100 µg/ml was chosen for in vitro subsequent labelling. No significant differences were found on Calcein AM/ethidium homodimer toxicity assessment between DIR at any concentration and matched controls from the same donor (N = 6) (Fig. 1b). Wound healing kinetics following standardized scratch wounding in HCEC's were similar between control and treated groups (Fig. 2). There was no significant difference in wound area remaining at any time point (n = 6, paired t-test) (Fig. 2d).
In vivo labelling of rabbit endothelial cells. Direct injection of DIR diluted in the rabbit aqueous to a final concentration of 100 µm/ml was successful in labelling rabbit endothelial cells. No adverse effects were observed following DIR injection, with no significant inflammation (conjunctival hyperemia, iris vascular dilation, anterior chamber cellular activity) seen at any time point. Slit lamp microscopy and infra-red imaging   (Fig. 3a). DIR stained endothelial cells were visible on infra-red imaging, with significant cell-to-cell variation in staining intensity observed (Fig. 3b). There was no increase in corneal thickness (Fig. 3c,f) at any time point after surgery, with CCT remaining < 400 µm throughout the observation period. At day 28, all corneas remained clear (Fig. 3d). DIR labelled cells were still visible, but with some reduction in observable fluorescence (Fig. 3e). DIR uptake was prominent in the iris, meaning that pupil dilation was necessary to reduce background fluorescence in subsequent examinations. Background fluorescence was further reduced by imaging using the higher magnification lenses, as these reduced the depth of focus (Fig. 3e).
In vivo corneal wounding: comparison between in vivo DIR imaging, confocal microscopy and immunohistology. DIR staining was compared to conventional confocal microscopy and post-sacrifice immunohistology in 6 rabbits (2 sacrificed at day 3, 2 at day 5 and 2 at day 28 post wounding). By using additional magnifying lenses attached to the HRA/Spectralis, it was possible to image individual DIR stained endothelial cells, although significant variability in the fluorescence of adjacent endothelial cells was observed (Fig. 4). During early wound healing (day 3), elongated, migrating endothelial cells were seen (Fig. 4a). This same morphology was observed on immunofluorescence staining performed after sacrifice of the same animal (Fig. 4d). At this stage, no endothelial imaging was possible with conventional confocal microscopy (Fig. 4g). There was  www.nature.com/scientificreports/ no difference in cell circularity measured from DIR and immunofluorescence images, with an average value of 0.51 ± 0.14 for histology derived circularity and 0.58 ± 0.12 for DIR imaging (t-test, p = 0.34). After wound closure, a confluent monolayer of cells with broad cell borders was seen with in vivo DIR imaging (Fig. 4b).
This corresponded to confluent cells with incomplete tight junction formation, as assessed by ZO-1 immunofluorescence staining (Fig. 4e). Conventional confocal imaging was still unable to produce useful images, largely because applanation of the cornea resulted in undulations of the posterior corneal surface (Fig. 4h). After initial wound closure, continuing maturation of central endothelial cells was observed. As time progressed, cell borders became narrower and cells became more circular (Fig. 4c). The regular endothelial mosaic observed with DIR staining at day 28 corresponded to small, largely hexagonal cells, with a continuous ring of ZO-1 staining seen on immunofluorescence (Fig. 4f). Conventional in-vivo confocal images could be obtained from day 14 onward as seen at day 28 ( Fig. 4i). Analysis at day 28 showed no difference in cell circularity (0.71 vs 0.74, t-test, p = 0.87) or the number of hexagonal cells as assessed by histology and DIR imaging. (50.9% (histology) vs 49.1% (HRA/ Spectralis), chi-squared, p = 0.9).
In vivo corneal wounding: comparison between in vivo DIR imaging and trypan blue staining. Good agreement was seen between macroscopic, non-invasive DIR imaging and intracameral trypan blue staining, both at the time of wound creation (Fig. 5a,b) and at day 3 after wounding ( Fig. 5c,d). Importantly, it was possible to acquire these images through corneas that were thickened or irregular in shape, as observed on the simultaneously acquired OCT (Fig. 5e). Bland-Altman plots were produced to compare the area and circularity of wounds (Fig. 6). Good agreement was seen between both methods, with less than 2% difference in area seen in any sample (Fig. 6a). Circularity was within 4% for all samples (Fig. 6b).
In vivo tracking of migrating endothelial cells following endothelial wounding in the rabbit. The wounding of the rabbit corneal endothelium in vivo was followed for 14 days using infra-red ( Fig. 7ad), OCT ( Fig. 7e-h), and DIR imaging ( Fig. 7i-l) with their respective magnified region ( Fig. 7m-p). Complete closure of an 8 mm endothelial scrape wound occurred by day 5 (Fig. 7k-o). CCT (as assessed by OCT) returned to pre-wound levels by day 14 in all cases (Fig. 7h). These findings are in keeping with our previously published results 10 obtained from scrape wounding in unstained corneas. Taken together, these findings suggest that DIR staining does not have a deleterious impact on endothelial healing or migration in vivo.
Labelling of ex vivo human corneas. Double staining using Calcein AM and DIR allowed multiplexed image acquisition of donor human corneas within the donor viewing chamber (Fig. 8a,b), as well as immediately post operatively inside the rabbit eye (Fig. 8c,d). Initially, staining with DIR showed large degrees of variation, with adjacent areas of the corneal endothelium showing markedly different degrees of fluorescence (Fig. 8b,d, yellow arrows). Calcein AM staining was able to detect large areas of cell loss (Fig. 8c, blue arrow) and differenti-   www.nature.com/scientificreports/ cell migration from the donor to host could be seen on DIR imaging. In the case illustrated, this resulted in loss of the straight edge of the triangular orientation mark (yellow arrows). Following animal sacrifice, endothelial cells were stained with anti-human nuclear antibodies (Fig. 9q,s, FITC channel). Migration of human endothelial cells (stained green) across the graft border was visible (Fig. 9q) and the pattern of donor cell distribution showed good concordance between DIR imaging and immunohistological assessment (Fig. 9p,q). Transfer of fluorescence to neighboring rabbit endothelial cells was not observed. Areas of rabbit endothelial migration onto the host DM were seen in DMEK cases (Fig. 9p,r) but not in DSAEK. These cells were distinguishable, by the lack of staining with both DIR (Fig. 9p,r) and anti-human nuclear antibodies (Fig. 9q,s). In one animal that had DMEK, clinical features of rejection (corneal oedema, ciliary injection) was observed at day 28 ( Fig. 10). Compared to day 21 images (Fig. 10a,b), DIR imaging showed central loss of fluorescence at lower magnification (Fig. 10c) and patchy attachment of small, circular endothelial cells at higher powers (Fig. 10d). This was associated with the onset of circulating, DIR positive, cells in the anterior chamber (Video S1). An anterior chamber tap confirmed the presence of cellular material, but the yield was too low to confirm cell origin or DIR positivity using FACS/immunofluorescence. High resolution cell imaging. By attaching the Rostock module (Heidelberg Engineering, Heidelberg, Germany) to the HRA/Spectralis, we were able to acquire detailed, high-resolution in vivo images. This allowed us to observe subtle biological processes at a cellular level, including nuclear fragmentation and cell detachment in both rabbit (Fig. 11a,b) and human (Fig. 11c) corneal endothelium; podocyte extension from neighboring cells (Fig. 11d); nuclei in anaphase (Fig. 11e) and daughter cell formation (Fig. 11f).

Discussion
Tracking of CECs in vivo is not widely performed to date due to a lack of suitable imaging techniques and paucity of data on labelling efficiency, duration of fluorescence, general toxicity, and effects of labelling on functionality of transplanted tissue 21 . The aim of this study was to determine whether NIR dye, and specifically for this study, DIR, could be used to label and track CECs, in vivo, without impacting cell viability or cellular function. We began by assessing the effect of DIR staining on wound healing, cell migration and cell viability, in vitro, using cultured primary human CEC's. At the concentrations required for in vivo imaging, DIR stained CECs migrated at the same rate as unlabeled CECs. At 24 h post-labelling, there was no significant difference in cell death between labelled and unlabeled cells. Taken together, these results suggest that DIR was a viable candidate for use as a tracker in CECs. We subsequently assessed whether use of DIR had any deleterious effects in vivo. A single intracameral DIR injection was sufficient to label rabbit endothelial cells and was not associated with any apparent inflammation or acute disturbance of corneal endothelial function. We subsequently used DIR imaging to track the endothelial cells on EK transplants from human tissue. We found it was possible to obtain high quality images of human endothelial cells within the anterior chamber following EK. To our knowledge, At the macroscopic level, we found DIR imaging to be comparable to repeated trypan blue injection when assessing endothelial wound healing following standard injury; a model commonly used to assess pharmacological therapies aimed at enhancing endothelial regeneration [22][23][24] . High magnification assessment of the healing edge of the wound showed that comparable morphological information could be obtained from in vivo imaging as compared to immuno-histology following animal sacrifice. A temporal relationship between closure of endothelial wounds, as observed by DIR imaging, and restoration of corneal clarity/CCT was also seen in all cases. In vivo wound healing kinetics were similar to those we previously reported in unlabeled cells 10 . Images could be acquired through opaque/edematous corneas, something not possible with conventional confocal imaging. Combined, these findings suggest that DIR staining has no significant effect on endothelial function or healing and allows continuous, in vivo, assessment at all time points post-wounding. Assessment of endothelial transplants stained with DIR showed no diffusion of DIR across cell membranes from adjacent endothelial cells, meaning that staining of rabbit endothelial cells did not occur following xenotransplantation with human DMEK grafts (Fig. 8). This allowed for a clear distinction between transplanted human endothelial cells and native rabbit endothelial cells, even if cells had migrated on or off the transplant itself. In vivo tracking of endothelial cells at pan-corneal and single-cell level. Images taken from an individual, DIR stained rabbit cornea. Following standard endothelial wounding, infra-red (a-d), OCT (e-h) and DIR images (i-p) were acquired using the HRA/Spectralis. Use of the anterior segment lens supplied by the manufacturer allowed macroscopic imaging of the whole cornea. By coupling additional lenses to the infinity optics retinal lens, higher magnification imaged could be captured (m-p). The images in the bottom row correspond to the areas seen in the white square outline in the macroscopic images. By sequentially imaging with increasing magnification, it was possible to track areas of interest. Immediately after scraping no DIR positive cells are seen in the wound area (i,m). At day 3 the cornea is still significant thicker that prewounding (f). The wound area can be seen to be much smaller (j, white box). At higher magnification levels, migrating cells closing the wound at the edge are seen (n). By day 5, although some residual corneal oedema and thickening persists on OCT imaging (g), complete wound closure can be observed (k,o). By day 14, corneal thickness has returned to normal (h) and a stable layer of DIR positive cells was observed with macroscopic (i) and higher magnification imaging (p). We have previously shown that rabbit endothelial cells are capable of rapidly migrating over bare DM with subsequent restoration of corneal clarity and thickness to pre-transplantation levels 10 . As such, it is important to be certain that any restoration of function is attributable to the transplanted cells and not a consequence of cellular regeneration in models where in vivo endothelial replication occurs 25 . Until now, detailed assessment of wound healing at different time points has relied either on repeated surgical interventions, in the case of trypan blue injection 10 , or on the sacrifice of animals at multiple time points 26 . Our assessments showed that a DIR fluorescence signal, allowing for high quality image acquisition, was retained for at least 28 days and in subsequent experimentation we have found fluorescence to be retained up to 60 days (data not shown).
With high resolution imaging, biological processes typically observed on histological examination could be seen in vivo; including cell spreading, apoptosis and cell division. In one animal, a reduction in corneal clarity, together with a development of corneal edema and an increase in corneal thickness occurred between days 21 and 28. DIR imaging showed numerous endothelial cells had detached from the DM and were freely floating in the aqueous. The cells remaining attached to DM displayed features consistent with an acute insult 13 . In the setting of xenotransplantation, these features were consistent with acute graft rejection 27,28 . In keeping with this, small DIR positive cells, consistent with previous reports of inflammatory cells, were observed interspersed with the endothelial cells were seen in both in the rabbit endothelial monolayer and transplanted human endothelial cells 29 .
Graft rejection remains a leading cause of transplant failure. Currently, models of rejection rely on indirect measures of endothelial function, and it is not always possible to separate rejection from other forms of endothelial trauma 11,30 . Methods of imaging inflammatory cell changes that proceed acute rejection in vivo will allow a greater understanding of the mechanisms at play 31 . Being able to assess the degree of cell death occurring during a rejection episode will provide information on the likelihood of recovery following rejection and effectiveness of anti-rejection strategies.
The use of NIR markers for tracking has several advantages. These tracers benefit from very low autofluorescence at NIR wavelengths and have excellent tissue penetration. This combination gives a high signal-to-noise ratio and allows non-invasive tracking of cells through the skin in organs such as the spleen or heart 32 . NIR tracers have recently been used in surgical trials to aid tumor clearance 33 and in clinical ophthalmic trials to assess cell loss in patients with early glaucoma 34 . Using an 800 nm band pass, sufficient emitted light is captured for high quality imaging, and this is possible even through opaque corneas with significant edema. The NIR dye, indocyanine green (ICG), has been used in ophthalmology for over 5 decades. This means that dedicated equipment for imaging of NIR tracers in the eye are widely available in clinical and research settings. Our imaging protocol uses clinical equipment, designed for ICG imaging, with minimal adjustments.
Given that DIR staining is a simple, non-toxic, one step process, it is possible that this method could be applied to graft tissue destined for human use if a good manufacturing practice form of the dye were made available. For organ cultured tissue, the point at which graft tissue is transferred to dextran containing media may provide an ideal opportunity for this 35 . It is possible that DIR may also be useful to track other cells used in corneal regenerative therapy such as limbal epithelial 21 or stromal stem cells 36 .
Our study showed that DIR fluorescence was initially quite heterogenous. This meant multiplexing with the viability dye, Calcein AM, was needed to assess immediate post-op graft viability. Fluorescence peaked at 24 h post transplantation making imaging easier, although some heterogeneity remained, meaning appropriately exposing the images was challenging. It may be possible to improve the use of DIR by combining several images taken at different sensitivities (iso settings); high dynamic range imaging 37 .
We believe that DIR imaging provides a powerful new tool for studying CECs in vivo. More detailed information about endothelial cell behavior following transplantation will improve our understanding of phenomena such as accelerated cell loss in high risk grafts and may lead to targeted therapeutics and improved graft survival. Its use in a research setting should ultimately lead to a reduction in the number of research animals needed and there is a potential for easy adoption in the clinical setting. Further studies confirming the exact length of time that DIR fluorescence persists in-vivo are warranted. , with informed consent from the next of kin. The isolation and subsequent propagation of the primary CECs was achieved using a dual media approach as described 38,39 . Briefly the DM is peeled from the donor cornea, and subsequently subjected to a two-step enzymatic treatment to release the CECs from the DM. Isolated cells were seeded onto pre-coated collagen culture plate at a seeding density of at least 1.0 × 10 4 cells/ cm 2 and established in a maintenance medium (M5-Endo; Human Endothelial-SFM supplemented with 5% serum) overnight. Subsequently, CECs were cultured in the proliferative medium (M4-F99; Ham's F12/M199, 5% serum, 20 μg/ml ascorbic acid, 1× ITS, and 10 ng/ml HrFGF) until they are approximately 80% confluent, before being re-exposed to M5-Endo for at least two days. The expanded CECs were then sub-cultured via single-cell dissociation using TrypLE Select, and plated at the desired seeding density for optimization studies. All cultured were carried out in a humidified atmosphere at 37 °C and 5% CO 2 .

Methods
For transplantation study, human corneo-scleral buttons with consent for research use were obtained from Miracles in Sight (Winston Salem, North Carolina, USA). Tissue preparation and transplantation was performed  40 , and all experiments involving these animals were approved by the Singhealth Institutional Animal Care and Use Committee (IACUC; 2016/SHS/1212), Singapore. Interventions were performed in New Zealand white rabbits aged 16-20 weeks, weighing between 3 and 3.5 kg. All surgical procedures and clinical evaluations were performed in a single eye of each rabbit under general anesthesia from intra-muscular xylazine 5 mg/kg (Troy Laboratories, Smithfield, Australia) and ketamine 40 mg/kg (Parnell Laboratories, Alexandria, Australia) with additional topical anesthesia (lidocaine 1%, Bausch and Lomb, Rochester) as previously described 10 .
Animals underwent phacoemulsification at least 1 week prior to endothelial keratoplasty to deepen the anterior chamber. Vancomycin was used in the irrigating fluid (20 µg/ml) for surgical procedures and rabbits received subconjunctival dexamethasone (1 mg) and gentamcin (0.4 mg) at the end of all surgical procedures.
Prior to EK, rabbits were injected with intravenous unfractionated heparin (1000 units in 1 ml of normal saline). Heparin was also added to the balanced salt solution used to irrigate the eye at a concentration of 50 units/ml. For all experiments, rabbits were examined daily for the first 5 days and then on days 7, 14, 21, and 28. Examinations consisted of slit lamp biomicroscopy, tonometry, optical coherence tomography, confocal fluorescence imaging using the Heidelbreg HRA/Spectralis (Heidelberg Engineering GmbH, Heidelberg, Germany) and conventional corneal confocal imaging using the Heidelberg HRT-II with the Rostock module (Heidelberg Engineering GmbH, Heidelberg, Germany).
Primary cultured CECs were seeded at a concentration of 2 × 10 5 cells/cm 2 in a Greiner 48-well flat bottom polystyrol microplate (Greiner, Frickenhausen, Germany), pre-coated with FNC coating mix. Cells were incubated with DIR diluted in M5 media 39 at a concentration of 10-200 µg/ml overnight at 37 °C (N = 15 per concentration). The following day, cells were washed thrice in fresh culture media and imaged using the HRA/ Spectralis at a standardized sensitivity of 100. Images were exported to ImageJ (National Institute of Health, Bethesda, USA) and converted to 8-bit. Mean grey values were measured in three separate regions of each plate and average fluorescence for each dose calculated. A comparison between groups was made using ANOVA.

Toxicity of DIR in vitro.
DIR-labelled endothelial cells and controls were cultured for a further 48 h in fresh media. Calcein AM/ethidium homodimer/Hoechst were added to the cultured cells to achieve final concentrations of 2 µM, 4 µM and 10 µM respectively. The plate was gently agitated, incubated at 37 °C for 30 min and imaged on the fluorescence microscope. The viable ECD was calculated and compared between control and treated cells. A positive control was performed by using the same viability assessment in cells incubated with 70% ethanol for 1 min prior to assessment. A comparison between groups was made using ANOVA.
Effect of DIR on endothelial cell migration. As the primary aim of DIR tracking was to examine cell migration, a scratch wound assay was performed. Primary cultured CEC's from the same donors were seeded onto different wells within the same plate. After reaching confluence, cells were cultured in M5 culture media with DIR 100 µg/ml or M5 media alone (controls). Each well received a single scratch using the tip of a 10 µl pipette. Plates were placed within the incuCyte imaging incubator (Sartorius, Essen Bioscience, Ann Arbor, Figure 8. Labelling of ex vivo human corneas. Human corneas were dual stained with Calcein AM and DIR dyes. (a) After preparation for implantation, the DMEK graft, resting on stroma, is returned to the viewing chamber and imaged using the FFA filter set to detect Calcein AM stained cells. Note the presence of a triangular orientation mark. Uniform green fluorescence is seen as areas of healthy cells and black areas represent dead or absent cells. (b) The same transplant is imaged using the ICG filters to detect DIR staining. There is marked variation in fluorescence across the graft meaning portions become either over or under exposed on global imaging. The yellow arrow points to an area of high DIR fluorescence. Comparison with the Calcein AM staining shows that most of the graft, including this area is cover by viable cells. (c) Global Calcien AM imaging of the graft, in vivo, immediately following transplantation shows a new area of cell death attributable to implantation trauma (blue arrow) (d) Global DIR imaging of the graft, in vivo, immediately following transplantation shows the same variation of fluorescence (e) High magnification imaging acquired by coupling a ×40 air objective microscope lens to the retinal imaging lens of the HFA/Spectralis. Individual Calcein AM negative cells are seen. (d) DIR imaging using the same lens shows DIR fluorescence in the cells that appeared non-fluorescent on the global image, suggesting the level of fluorescence is variable, but present across the entire graft. At this time point, comparison between DIR and Calcein AM imaging shows DIR does not differentiate between living and dead, but attached, cells. www.nature.com/scientificreports/ Michigan, USA). The incuCyte is a multiplex imaging system that is incorporated into an incubator, allowing continuous imaging in multiple wells without alterations in temperature or atmosphere. Image registration to define the scratch area was performed and serial phase-contrast images were taken every hour for 24 h. The wound area was measured every 3 h and expressed as a percentage of that measured at T = 0 h. Wound size was compared between stained cells and unstained controls using multiple t-tests. Imaging of endothelial cells post-operatively. Imaging was performed using the HRA/Spectralis set in the ICG mode. In the ICG mode, the HRA/Spectralis utilizes a 790 nm diode excitation laser and 800 nm long-pass filter. Images were captured at varying levels of magnification by attaching microscope objective lenses (Nikon, Japan) or the Rostock module to the standard fundus imaging lens by using custom built collars, as previously described 3 . Use of multiple lenses allowed both macroscopic and individual cell level imaging.

Comparison of DIR imaging and established methods in a wound healing model. Two weeks
following DIR injection, 9 rabbits had 8 mm endothelial scrape wounds as described in our previous work 10 . 6 rabbits were sacrificed at day 3, one at day 5 and the remaining two animals were sacrificed at day 28. Pan-cor- www.nature.com/scientificreports/ neal images were acquired using the manufacture supplied anterior segment lens for the HRA/spectralis. These allowed tracking of the entire wound areas, macroscopically. For higher magnification imaging, the inferior edge of the wound was located by moving the lens array up from the inferior limbus until the leading edge of migrating cells was seen, allowing a consistent area to be sampled for DIR and conventional in vivo confocal microscopy. In rabbits sacrificed at day 3, the endothelial wound was assessed by injecting trypan blue into the anterior chamber as previously described 5 . Trypan blue and DIR images were exported to ImageJ and the wound outlined using the polygon selection tool. This was converted to a smoothed spline and measurements of wound area and circularity were taken. The measurements for imaging technique were compared using a Bland-Altman plots.
After killing, the inferior cornea was marked by passage of a 6-0 silk suture through the limbus at the 6 o' clock position and used as a reference for immunohistology.
The corneas were harvested, washed in BSS and fixed in 1% paraformaldehyde at room temperature for 5 min. Samples were permeabilized in Triton-X 1% for 5 min and then blocked using and 2 step process, first in 10% goat serum for 45 min followed by 3% BSA for 45 min. Samples were incubated with primary antibody to ZO-1 (1:300, Thermofischer) or Human Nuclear Antigen (1:100, Merck-Millipore) at 4 °C for 24 h in a solution composed of phosphate buffered saline (PBS) with 0.1% Triton-X and 1% BSA. Samples were washed 3× with PBS, each for 10 min, and then incubated with secondary antibody, Hoechst (1:1000) and phalloidin (1:500), as required, overnight at 4 °C. To assess specificity of the immunostaining, corneas were processed without primary antibody. Specimens were washed 3× in PBS, re-fixed in 3% PFA, washed again and flat mounted in ProLong gold. The entire cornea was flat mounted and the inferior edge of the wounded area, in line with the silk suture www.nature.com/scientificreports/ was identified for imaging. Individual cells were manually outlined and cell metrics compared between in vivo images and flat-mounted, immunofluorescence images (stained with phalloidin, Hoechst and ZO-1). At day 3, when cells were still migrating, a comparison between cell circularity was made (a minimum of 100 cells were measured in each group). At day 28 when a stable monolayer was present, a comparison between cell circularity and the percentage of hexagon cells was made (neighbor analysis function in the BioVoxxel toolbox for ImageJ).
Labelling of ex vivo human corneas. Human corneas were labelled prior to tissue preparation for Descemet Membrane Endothelial Keratoplasty (DMEK) and following mircokeratome cutting for Descemet Stripping Automated Endothelial Keratoplasty (DSAEK). The procedure was optimized in a series of initial experiments. To limit the amount of dye required and prevent corneal swelling, the corneas were kept within the Optisol-filled viewing chamber. Optisol was aspirated from the corneal cup and the endothelial surface further dried with cellulose sponges. DIR (100 µg/ml), diluted in M5 medium, was pipetted onto the endothelial surface until the entire endothelium and trabecular meshwork were covered. The epithelial surface remained suspended by the pillars of the viewing chamber and in contact with the Optisol, with the scleral rim acting as barrier to prevent the two media for mixing. The lid for the viewing chamber was replaced loosely and the corneas were incubated over night at 37 °C. For corneas in which a comparison between DIR and Calcein AM fluorescence was performed, corneas were rinsed 3× in fresh M5 media and the incubated with Calcein AM 1.67 µM in BSS for 30 min prior to surgery. Tissue was prepared and implanted using our previously described techniques for DMEK (n = 4) 5,41 and DSAEK (n = 6) 42 and imaging for Calcein AM and DIR fluorescence performed alongside the standard examinations, following the review schedule outlined above.
Statistical analysis. All statistical analysis was conducted using Prism (Graphpad software LLC, San Diego, USA). Unless otherwise stated significance was set at 5%. For multiple comparisons a Bonferroni adjustment was performed.